1. Field of the Invention
The present invention relates to identifying genes, particularly expressed genes in microorgansims.
2. Description of Related Art
There is often a need to identify expressed genes in soil particularly in contaminated soil so as to counter or reduce the effects of contaminates therein. Methods for the isolation and quantification of mRNA from environmental samples are designed to specifically measure in situ gene expression and activity. Direct extraction of mRNA from soil or soils and quantification of mRNA by ribonuclease protection assay has been demonstrated for naphthalene dioxygenase in soils and sMMO in aquifer sediments. Reverse transcriptase PCR amplification of mRNA for sMMO in activated sludge and lignin peroxidase in soils has also been demonstrated. These methods for mRNA analysis are a natural compliment to DNA extraction and hybridization or PCR analysis to detect gene sequences for catabolic genes or rDNA gene abundance in natural samples. In addition, direct mRNA analysis also provides potential evidence of in situ gene activity and conditions permissive of gene induction in the environment. These applications can be independent of microbial cultivation and hence, avoid many laboratory biases. However, these previous mRNA analytical methods are limited by the need for a priori information on gene sequences in order to design specific probes or primers for mRNA measurement. And there is a need to explore the use of differential display (DD) to quantify and recover novel mRNAs and/or cryptic or unknown DNA sequences transcribed under in situ conditions in soil.
For further information on DD see U.S. Pat. No. 5, 665,547 to Pardee et al (1997) incorporated herein by reference.
While DD has very recently been applied to environmentally related research, the focus has been limited to eukaryotes. The technique has been used to discover genes induced in white-rot fungus by pentachlorophenol and rat Sertoli cells by cadmium acetate and polychlorinated biphenyls. In this regard, DD potentially allows identification of known or cryptic microbial genes that are differentially expressed under altered field conditions, such as chemical exposure, oxidative stress, extreme pH, anaerobiosis, heat shock, and starvation.
DD and the closely related RAP-PCR have been used to detect and isolate differentially expressed genes under induced and uninduced conditions in both eukaryotes and prokaryotes. The DD procedure, which uses a poly T primer for the RT reaction and an additional arbitrary primer for PCR has been exclusively applied to eukaryotic expression studies. RAP-PCR differs from DD in that arbitrary primers are used for both the RT and PCR steps, and as such, has been used for both eukaryotic and prokaryotic studies. For a further discussion of RAP-PCR see Welsh J. et al, Arbitrarily Primed PCR Fingerprinting of RNA, Nucleic Acids Res., Vol 20; 4965-4970, which is incorporated herein by reference.
Accordingly, there is need and market to detect and isolate differentially expressed genes under induced and uninduced conditions in microorganisms such as cultures including pure cultures and soil microcosms. And there has now been discovered such a method per the present invention.